Recent data from our laboratory and others suggest that the developing brain responds differently to hypoxia-ischemia than the mature brain, therefore requiring different strategies for neuroprotection. The ability of the cell to survive depends upon which downstream regulators will be able to provide the appropriate defense mechanisms. One such protective mechanism is the transcription factor hypoxia inducible factor, HIF1. In previous studies we have shown that HIF1a is upregulated to cause tolerance to hypoxic stress. HIF1 upregulates a number of target genes that allow the cell to survive hypoxic stress and maintain oxygen homeostasis. In this proposal, we will use a new model that we developed during the last cycle of this grant to test the following hypothesis. It is our hypothesis that HIF1a induction after hypoxia and ischemia leads to upregulation of survival genes resulting in neuroprotection in the developing nervous system. We will use the transient focal ischemia-reperfusion and hypoxia-ischemia models in neonatal rats and mice to explore the role of HIF1a in neuroprotection. The focal ischemia model allows us to determine the response of expression of HIF1a in vivo to varying degrees of ischemia. We will determine histological and neurobehavioral outcome and target gene expression correlating with HIF1a mRNA and protein expression as we vary levels of ischemia and monitor infarct evolution using MRI. We will then determine whether ERK phosphorylation is a necessary step in transcriptional activity of HIF and whether it is required for neuroprotection. During the last cycle of this grant, Dr. Holtzman showed that BDNF worked through ERK signaling to provide neuroprotection after neonatal HI. We will modulate HIF1 signaling with ERK inhibitors to determine if these signaling pathways are intact. We will then determine the role of desferrioxamine and cobalt chloride in HIF1 activation in regard to neuroprotection. In this way we can investigate both hypoxia dependent and independent mechanisms of HIF1. We will use cre/lox knockout mice and perform hypoxia-ischemia to better define cell death pathways in the absence of HIF1. In cultured neurons, we will explore the mechanisms of HIF1 stabilization and phosphorylation in cultured neurons in regard to cell type specificity. This information will be incorporated into the information of Projects 3 and 4 and will inform the human studies in Project 1. This project makes extensive use of the imaging and behavioral cores.